A novel preparation of dissociated renal proximal tubule cells that maintain epithelial polarity in suspension.
A novel preparation of dissociated renal proximal tubule cells that maintain epithelial polarity in suspension.
复制标题
一种分离的肾近端小管细胞的新型制剂，可在悬浮液中维持上皮极性。
DOI：
10.1152/ajpcell.1996.270.6.c1843
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Maunsbach,AB
中科院分区：
文献类型：
--
作者：
Segal,AS;Boulpaep,EL;Maunsbach,AB
上皮细胞的功能特性与其极化结构密不可分。在单个细胞水平上研究极性是困难的，因为大多数上皮细胞在解离后几分钟内就失去了极性。我们已经开发了一种制备天然的，解离的，钝口螈近端小管细胞，保持结构和功能极性的至少7天的悬浮液。我们已经使用这些细胞来探索单个细胞中的细胞表面极性。电镜细胞化学和免疫细胞化学研究表明碱性磷酸酶仅定位于顶端刷状缘，而Na（+）-K（+）-ATP酶仅限于基底外侧膜。正如在近端小管原位，尖锐的结构之间的过渡顶端和基底侧膜域被保留。在原位紧密连接处发现的ZO-1蛋白不存在于解离细胞的膜上，而是分布在细胞质中。肌动蛋白细胞骨架在单个细胞中也保持极化，其分布和组织似乎有助于维持细胞极性。电生理测量表明，这些细胞保持活力，至少只要他们保持结构极化。膜片钳记录从顶部和基底侧膜显示，几个离子通道蛋白的分布保持功能极性。我们推测，尽管损失的细胞间的“门”和膜相关的ZO-1，所谓的“栅栏”功能的紧密连接复合物保留在这些解离的近端小管细胞。该制剂可作为一种有用的单细胞模型，用于研究上皮极性和膜运输途径。
The functional properties of an epithelium are inextricably linked to its polarized structure. It has been difficult to study polarity at the level of the single cell, since most epithelial cells lose their polarity within minutes after dissociation. We have developed a preparation of native, dissociated, Ambystoma proximal tubule cells that maintain structural and functional polarity for a minimum of 7 days in suspension. We have used these cells to explore cell surface polarity in a single cell. Electron microscope cytochemical and immunocytochemical studies show that alkaline phosphatase is localized exclusively to the apical brush border, whereas the Na(+)-K(+)-ATPase is restricted to the basolateral membrane. Just as in the proximal tubule in situ, a sharp structural transition between the apical and basolateral membrane domains is retained. The ZO-1 protein found at the tight junction in situ is not present on the membrane of the dissociated cells, but rather it is distributed in the cytoplasm. The actin cytoskeleton also remains polarized in the single cells, and its distribution and organization appear to help maintain cell polarity. Electrophysiological measurements show that these cells remain viable at least as long as they remain structurally polarized. Patch-clamp recordings from both the apical and basolateral membranes show that the distribution of several ion channel proteins maintains functional polarity. We hypothesize that, despite loss of the intercellular “gate” and membrane-associated ZO-1, the socalled “fence” function of the tight junctional complex is retained in these dissociated proximal tubule cells. This preparation may serve as a useful single cell model with which to study epithelial polarity and membrane trafficking pathways.